From June 21st to 24th, the annual international liver disease conference, the 58th Annual Meeting of the European Association for the Study of the Liver (EASL 2023) and EASL Congress 2023, was grandly held in Vienna, Austria. This year, over a hundred research abstracts from Chinese experts and scholars were selected for presentation at the conference. Among them, teams from Peking University People’s Hospital and Peking University Hepatology Institute presented seven posters in the fields of non-alcoholic fatty liver disease (NAFLD), liver cancer, chronic hepatitis B, and primary biliary cholangitis. Below, we provide details on these research abstracts, giving you a glimpse of the teams’ achievements.
1. Relationship Between Multiple Vitamin Co-Exposure and Mortality Risk in Metabolic – associated Fatty Liver Disease (MAFLD)
Background: Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease associated with insulin resistance and genetic susceptibility, affecting approximately one-fourth of the global population and posing significant public health challenges. Numerous studies have explored the relationship between vitamins and MAFLD, suggesting that vitamin levels may impact disease progression outcomes, but the results remain controversial. Therefore, the impact of multiple vitamin co-exposure on MAFLD deserves further investigation.
Method: Data from the 1988-1994 National Health and Nutrition Examination Survey (NHANES-III) and its associated mortality follow-up were utilized. MAFLD was defined as ultrasound-diagnosed liver fat deposition and the presence of at least one of the following conditions: overweight or obesity, type 2 diabetes, or metabolic dysfunction. K-means clustering determined patterns of multiple vitamin co-exposure. Logistic regression models were used to analyze disease risk (OR), and Cox proportional hazards models were employed to analyze all-cause and specific mortality risks (HR).
Results: Multivariate logistic regression analysis showed that higher vitamin levels were associated with a reduced risk of MAFLD. Higher vitamin levels were associated with a reduced all-cause mortality rate in the overall population. Higher levels of vitamin A and vitamin D were associated with reduced all-cause mortality in MAFLD patients, and higher vitamin D levels were associated with reduced cardiovascular and cancer mortality rates in MAFLD patients.
Conclusion: Higher levels of multiple vitamin co-exposure may moderately reduce the risk of MAFLD, and higher vitamin D levels may reduce the overall, cardiovascular, and cancer mortality risks in MAFLD patients.
2. IP10 and Anti-HBc Predict Virological Relapse and HBsAg Loss for Chronic Hepatitis B Patients After Nucleos(t)ide Analogue Discontinuation
Background: The risk of virological relapse (VR) and the possibility of achieving HBsAg seroclearance after discontinuation of nucleos(t)ide analogue (NA) therapy in HBeAg-positive chronic hepatitis B (CHB) patients have been topics of controversy. This study aimed to evaluate the predictive ability of interferon-induced protein 10 (IP10) and anti-HBc for VR and HBsAg seroclearance after NA discontinuation in these patients.
Method: From January 2017 to December 2020, 139 HBeAg-positive CHB patients who met the discontinuation criteria according to the Chinese Chronic Hepatitis B Prevention and Treatment Guidelines (2015 version) were prospectively enrolled from 12 hospitals in the Beijing-Tianjin-Hebei region. Patients were followed up every 3 months after NA discontinuation until 24 months or clinical relapse. Clinical data were collected, clinical specimens were obtained, and liver tissue samples were analyzed for VR rates and HBsAg seroclearance rates. IP10 and anti-HBc’s predictive abilities were evaluated.
Results: At the end of treatment (EOT), the IP10 and anti-HBc levels were 29.2 (5.1-66.4) pg/mL and 193.6 (136.9-221.4) IU/mL, respectively. Cox regression analysis results showed that EOT IP10 (HR 0.976, P < 0.001) and anti-HBc (HR 1.011, P < 0.001) were independent predictors of VR after discontinuation. EOT IP10 (HR 1.026, P < 0.001) was also an independent predictor of HBsAg seroclearance after discontinuation.
At 24 months after discontinuation, the cumulative VR rates for patients with EOT IP10 > 26.99 pg/mL and ≤ 26.99 pg/mL were 31.9% and 70.1%, respectively (HR 2.998, P < 0.001). The cumulative VR rates for patients with EOT anti-HBc ≤ 193.6 IU/mL and > 193.6 IU/mL were 34.8% and 64.6%, respectively (HR 2.213, P = 0.005).
Conclusion: EOT IP10 and anti-HBc levels can predict VR and HBsAg seroclearance after NA discontinuation in HBeAg-positive CHB patients, providing potential guidance for clinical decision-making.
3. Collagen co-localized with macrovesicular steatosis better differentiates fibrosis progression in non-alcoholic fatty liver disease mouse models
Background: Non-alcoholic fatty liver disease (NAFLD) is a global commonly occurring liver disease. However, its exact pathogenesis is not fully understood. The purpose of this study was to quantitatively evaluate the progression of steatosis and fibrosis by examining their distribution, morphology, and co-localization in NAFLD animal models
Method: Six mouse NAFLD groups were established: (1) western diet (WD) group; (2) WD with fructose in drinking water (WDF) group; (3) WDF + carbon tetrachloride (CCl4) group, WDF plus intraperitoneal injection of CCl4; (4) high-fat diet (HFD) group, (5) HFD with fructose (HFDF) group; and (6) HFDF + CCl4 group, HFDF plus intraperitoneal injection of CCl4. Liver tissue specimens from NAFLD model mice were collected at different time points. All the tissues were serially sectioned for histological staining and Second-harmonic generation (SHG)/two-photon excitation fluorescence imaging (TPEF) imaging. The progression of steatosis and fibrosis was analyzed using SHG/TPEF quantitative parameters with respect to the NASH CRN scoring system
Results: qSteatosis showed a good correlation with steatosis grade (R: 0.823–0.953, P < 0.05) and demonstrated high performance (AUC: 0.617-1) in six mouse models. Based on their high correlation with histological scoring, qFibrosis containing four shared parameters (#LongStrPS, #ThinStrPS, #ThinStrPSAgg, and #LongStrPSDis) were selected to create a linear model that could accurately identify differences among fibrosis stages (AUC: 0.725-1). qFibrosis colocalized with macrosteatosis generally correlated better with histological scoring and had a higher AUC in six animal models (AUC: 0.846-1).
Conclusion: Quantitative assessment using SHG/TPEF technology can be used to monitor different types of steatoses and fibrosis progression in NAFLD models. The collagen co-localized with macrosteatosis could better differentiate fibrosis progression and might aid in developing a more reliable and translatable fibrosis evaluation tool for animal models of NAFLD.
4. PSMP inhibits HCC progression by regulating the polarization of tumor-associated macrophages via the PI3K/Akt pathway
Background: Hepatocellular carcinoma (HCC) is one of the malignant tumors with high morbidity and high mortality, which is prone to metastasis and recurrence and has a poor prognosis. PC3 secreted microprotein (PSMP) or microprotamine (MSMP) is a novel chemotactic cytokine discovered through genome-wide bioinformatics analysis and chemoattractant platform screening, which can act as a CCR2 ligand to recruit peripheral blood monocytes and lymphocytes. Our previous study found that PSMP was significantly highly expressed in human and mouse liver fibrosis/cirrhosis tissues induced by different causes. PSMP can promote the progression of liver fibrosis by regulating the infiltration, activation and polarization of macrophages. However, the relationship between PSMP and the development and prognosis of HCC remains unclear.
Method: The expression of PSMP was detected in two independent HCC patients cohorts and its correlation with patients’ prognosis was analyzed. In vivo: In PSMP knockout and wild-type mice, two mouse HCC cell lines (Hepa1-6, H22) were used to construct the subcutaneous tumorigenesis models and the liver orthotopic tumorigenesis models; In nude mice, two human HCC cell lines (BEL-7402, BEL-7405) were used to construct the subcutaneous tumorigenesis models. The direct effects of PSMP on the polarization of macrophages (human THP-1 cell line and mouse bone marrow-derived macrophages (BMDMs) were studied in vitro. In addition, we performed RNA sequencing of BMDMs from WT and PSMP gene knockout mice.
Results: Through the detection of clinical HCC patient samples, we found that PSMP is downregulated in human HCC tissues, and its expression level is positively correlated with the prognosis of HCC patients. In vivo, we found that knockout of PSMP promotes subcutaneous and liver orthotopic tumor growth in mice; Overexpression of PSMP inhibits the formation of subcutaneous tumors in nude mice. Further, knockout of PSMP significantly inhibits the infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) and promotes the infiltration and polarization of M2-type tumorassociated macrophages (TAMs) in mice. In vitro, we found that PSMP could directly promote M1-polarization and inhibit M2-polarization of human THP-1 cells and mouse BMDMs. In addition, analysis of RNA sequencing results showed that PSMP may mediate the polarization of macrophages by regulating the PI3K/Akt signaling pathway.
Conclusion: Taken together, PSMP may inhibit the M2 polarization of tumor-associated macrophages through the PI3K/Akt signaling pathway, and then promote the anti-tumor immune response of CD8+ T cells, and ultimately inhibit the progression of HCC. The results are expected to clarify the role and mechanism of PSMP in the liver tumor microenvironment for the first time, which has important theoretical significance, and may also provide new targets for the treatment of HCC, with potential application value.
5. The mutated ENTPD6 as neoantigen in hepatocellular carcinoma
Background: Tumor neoantigens, new peptides generated by somatic mutations from tumor tissues but not normal tissues, have good tumor specificity and strong immunogenicity. Though Clinical trials testing neoantigen immunotherapy have yielded encouraging results in patients with hepatocellular carcinoma (HCC) worldwide, it’s hard to know which neoantigens are dominant in determining the responsiveness to immunotherapy treatment. In this study, we used the Co-HA system, a single-plasmid system co-expressing patient HLA and antigen, to detect the immunogenicity of neoantigens and identify new dominant hepatocellular carcinoma (HCC) neoantigens..
Method: First, we enrolled 14 HCC patients for next-generation sequencing for variation calling and predicting potential neoantigens. Then, the specific cytotoxicity generated by this neoantigen was shown using the Co-HA system. Finally, potential HCC-dominant neoantigens were screened out by tetramer staining and validated by the Co-HA system using methods including flow cytometry, ELISPOT, ELISA and sequencing.
Results: First, 2875 somatic mutations were identified in 14 HCC patients. The main base substitutions were C > T/G > A transitions, and the main mutational signatures were 4, 1 and 16. The highfrequency mutated genes included HMCN1, TTN and TP53. Then, 541 potential neoantigens were predicted. Moreover, 37 predicted neoantigens restricted by HLA-A*11:01, HLA-A*24:02 or HLAA*02:01 were assayed by tetramer staining to screen out potential HCC-dominant neoantigens. Furthermore, the Co-HA system identified that the HLA-A*24:02-restricted epitope 5′-FYAFSCYYDL-3′, produced by the mutated ectonucleoside triphosphate diphosphohydrolase 6 (ENTPD6) had strong immunogenicity in HCC. Finally, Huh-7 cells co-expressing HLA-A*24:02 and ENTPD6 neoantigen were selected as the target cells and implanted into the B-NDGB2mtm1Fcrntm1 (mB2m)/Bcgen mouse model. When tumor size was approximately 150 ± 50 mm3, they were treated with the neoantigenspecific T cells through intratumor injection every 2 days. After 3 times of treatment, the volume and weight of tumors with neoantigens were both smaller than those of WT type..
Conclusion: The HLA-A*24:02-restricted epitope 5′-FYAFSCYYDL-3′, produced by the mutated ENTPD6, was the dominant neoantigen with high immunogenicity in HCC.
6. Lower ALT levels are associated with increased all-cause and cardiovascular mortality in patients with NAFLD in the United States population
Background: The serum alanine aminotransferase (ALT) level is often considered as a marker to evaluate the activity of liver disease and the severity of liver injury. In this study, we aimed to investigate the association between ALT levels and all-cause and cause-specific mortality in non-alcoholic fatty liver disease (NAFLD) patients.
Method: National Health and Nutrition Examination Survey (NHANES)-III from 1988 to 1994 and NHANES-III related mortality data from 2019 were used. NAFLD was defined as hepatic steatosis diagnosed by ultrasound, without other liver diseases. ALT levels were categorized into four groups according to different recommended upper limit of normal in men and women: <0.5 upper limit of normal (ULN), 0.5–1 ULN, 1–2 ULN, ≥2 ULN. The hazard ratios (HRs) for all-cause mortality and cause-specific mortality were analyzed with Cox proportional hazard model.
Results: Multivariate logistic regression analysis demonstrated odds ratio of NAFLD correlated positively with increasing serum ALT levels. In patients with NAFLD, all-cause mortality and cardiovascular mortality are the highest when ALT<0.5ULN, yet cancer-related mortality is the highest when ALT ≥2ULN. The same results could be found in both men and women. Univariate analysis showed that severe NAFLD with normal ALT level had the highest all-cause and cause-specific mortality, but the difference was not statistically significant after adjustment for age and multivariate factors.
Conclusion: The risk of NAFLD was positively correlated with ALT level, but all-cause and cardiovascular mortality were the highest when ALT<0.5ULN. Regardless of the severity of NAFLD, normal or lower ALT levels are associated with higher mortality than elevated ALT levels. Clinicians should be aware of not only high ALT, indicating liver injury, but also low ALT associated with higher risk of death.
7. Pyroptosis plays a key role in primary biliary cholangitis of humans and mice
Background: Primary biliary cholangitis (PBC) is a progressive, non-suppurative, destructive intrahepatic cholestatic disease, which is considered to be an immunological disorder with both environmental and genetical participation. Around 40% PBC patients were reported to have an incomplete response to the firstline treatment, ursodeoxycholic acid (UDCA), leading to a poor prognosis and lack efficient treatment. Pyroptosis, a newly discovered cell death pathway, characterized by gasdermin-mediated programmed necrosis, were proved to be involved in many liver diseases. However, if pyroptosis is involved in the pathogenesis of PBC remains unclear. In this study, we aim to investigate the involvement of pyroptosis in PBC patients and mice and explore the potential of pyroptotic pathway as a possible treatment target.
Method: Liver tissue samples were collected from liver biopsy of six PBC patients with no history of other liver diseases from Hepatology Department of Peking University People’s Hospital. Samples from control individuals with normal liver histology were collected from patients with no evidence of other liver diseases. Twenty female C57BL/6 mice of 4–6 months old were evenly divided into PBC group and control group. PBC mice were induced with two doses of 2-nonynoic acid (2OA-BSA) and polycytidylic acid (poly I: C) for total 12 weeks. Immunocyte type correlation analysis was performed on the GSE119600 dataset of Gene Expression Omnibus (GEO) database with whole blood samples from PBC patients (n = 90) and non-liver disease controls (n = 47). Immunohistochemistry (IHC) staining of gasdermin D (GSDMD) in liver samples of PBC patients and mice, and the expression levels of GSDMD and its classical pathway were determined in the PBC mice model.
Results: The immunocyte type correlation analysis reported that the expression of key transcription factors of M1 macrophages-NOS2, IRF5, PTGS2 were significantly higher in PBC patients than that of control group (p < 0.05). The expression levels of GSDMD in liver samples of PBC patients were significantly improved. Noteworthily, the IHC staining of GSDMD in the PBC liver sections showed a distribution pattern of macrophage. For the 2OAinduced PBC mice model, pyroptosis pathway was upregulated than control mice. Both the mRNA (p < 0.05) and protein levels (p < 0.05) of GSDMD significantly increased compared to normal controls, determined by qrt-PCR and western blotting, respectively. Besides, the qrt-PCR showed that the expression levels of all key elements of classical pyroptosis pathway (i.e., NLRP3-Caspase-1- GSDMD) was significantly higher than control (p < 0.05). IHC staining also revealed improved GSDMD and Caspase-1 expression in PBC mice, especially around the portal area.
Conclusion: Pyroptosis plays a key role in PBC patients and 2OA-BSA induced PBC mice, possibly macrophages being the most important executors. Inhibition of the pyroptosis pathway might be a potential target for the future treatment of PBC.
